1. Field of Invention
This invention relates to the chemical transformation of substances by living organisms, especially microorganisms. Products of such biotransformations range from foods such as yogurt and cheese to drugs such as steroidal hormones or drug intermediates such as L-phenylpropan-1-ol-2-one.
2. Discussion of Prior Art
All U.S. Patents cited herein are thereby incorporated herein by reference.
Production and accumulation of useful products formed by living organisms predates recorded history as in wine or beer production by fermentation. A comprehensive guide to compounds that are accumulated, produced, transformed or in some way acted upon by microorganisms and cell lines is Microbes and Cells at Work, published by American Type Culture Collection, Rockville, Md. (1988). Useful products so produced include antibiotics, amino acids, enzymes, organic fatty acids, nucleic acids, vaccines, vitamins, peptide hormones, steroid hormones, dextran and many other substances. A specific example is the use of Aspergillus species to transform progesterone to dihydroxyprogesterone.
Of specific interest for the present invention is the bioconversion of a substrate that is toxic to the living organism which brings about the biotransformation. Bioconversions of toxic substrates are very important in the treatment of toxic waste substances such as chlorinated hydrocarbons, pesticide residues such as organic phosphates or effluents from the manufacture of explosives such as dilute solutions of TNT or RDX which are very toxic compounds.
A specific case of biotransformation of a toxic substrate to a useful compound is the microbial biotransformation of benzaldehyde to L-1-ph-enylpropan-1-ol-2-one, frequently termed L-phenylacetyl carbinol and sometimes referred to herein as L-PAC. L-PAC is used as a raw material for the chemical synthesis of the drug ephedrine by a process described in U.S. Pat. No. 1,956,950 to Hildebrand and Klavehn (1934). Benzaldehyde is converted to L-PAC when added to a fermentation medium previously inoculated with yeast cell mass in the presence of fermentable sugar, a bioconversion reported as early as 1921 by Neuberg and Hirsch (Biochem Zeitschrift 115, 282-310) and Neuberg and Libermann, ibid 121, 311-339. See also Neuberg and Ohle ibid 127, 327-339 (1922) and 128, 610-618 (1922). The production of L-PAC from benzaldehyde by different species of yeast has been investigated by Gupta et al Biotechnol. & Bioeng. 21, 1085-89 (1979), Netrival et al [Eur. J. Appl. Microb. Biotech. 16, 35-38 (1982)] and Bringer-Meyer et al [ Biocatalysis 1, 321 (1988)]. Groger et al [Z. Fur Allgem. Mikrobial. 6(4), 275-87 (1966)], Voets et al [Z. fur Allgemm. Mikrobiol 13, 355-366 (1973)] and Agarwal et al [Biotechnol. and Bioeng. 29, 783-785 (1987) have investigated various processing methods and variables related to this bioprocess. Under normal fermentation conditions, quantitative conversion of benzaldehyde to L-PAC is never achieved, as reported by Smith et al [J. Bacterial 65, 440-45 (1953)] and Gupta et al (cited above).
The advantages of using immobilized cells for microbial biotransformation have been discussed by Arcuri et al [Biotechnol. & Bioeng. 25, 2399-2411 (1983)], Bihari et al [Biotechnol. & Bioeng. 26, 1403-08 (1984)], Scott [Enzyme & Microbial Tech. 9, 66-73 (1987)] and Fukui et al [Experientia 45, 1055-611 (1989)]. Fukui et al point out that a general disadvantage of immobilized living cells is undesirable metabolic activities which reduce product yields and increase permeability barriers for substrates and metabolites. Scott also cites the observation of Brodelius et al [Ann. N.Y. Acad. Sci. 434, 496-500 (1984)] that general microbial metabolism decreases upon immobilization, and the report of Robinson et al [Enzyme & Microbial Technology 7, 212-216 (1985)] that both respiration and growth rates of immobilized organisms decrease with increased cell concentration. Surprisingly, and in contrast to the findings of Fukui et al, Scott, Brodelins et al and Robinson et al we have found that immobilizing the yeast cells causes the yield of L-PAC from benzaldehyde to increase. In addition, we have also found that immobilization of the yeast cells also permits their exposure to higher concentrations of benzaldehyde while permitting higher productivity of L-PAC product from the biotransformation reaction. We hypothesize that immobilization may protect the yeast cells from toxic effects of the benzaldehyde substrate and perhaps also from toxic effects of the L-PAC product. This protection appears to occur in addition to the other well known favorable effects of cell immobilization, such as helping to maintain the cells in a bioreactor, facilitating the separation, recycling and re-use of the cells and protection of the cells from shear stress.
Bar [Appl. Microbiology and Biotechnology, 31, 225-28 (1989)] has reported that the presence of alpha-cyclodextrin (ACD) greatly enhances the conversion of benzylaldehyde to benzyl alcohol by yeast. Using an ACD concentration (27.5 g/L or 0.028 mols/l) which was greater than the molar equivalent of the benzaldehyde concentration (which was 3 g/L or 0.024 mol/L), Bar found that the yield of benzyl alcohol was increased to 67.4% (molar) after 24 hours, compared to only 15.4% (molar) in the absence of ACD.
The chemical equations representing the possible bioconversion reactions of benzaldehyde are: EQU C.sub.6 H.sub.5 CHO+H.sup.+ +NADH.fwdarw.C.sub.6 H.sub.5 CH.sub.2 OH+NAD.sup.+ A. EQU C.sub.6 H.sub.5 CHO+1/2 O.sub.2 .fwdarw.C.sub.6 H.sub.5 COOH B. EQU C.sub.6 H.sub.5 CHO+CH.sub.3 CHO.fwdarw.C.sub.6 H.sub.5 CHOHCHOCH.sub.3 C.
As explained by Bringer-Meyer et al [Biocatalysis 1, 321-31 (1988)] acetaldehyde, which reacts with benzaldehyde to produce L-PAC according to reaction C above, is formed by the decomposition of pyruvate in yeast by pyruvate decarboxylase. It is evident that the greatly increased yield of benzyl alcohol to 67% (molar) by reaction A as observed by Bar must be associated with greatly reduced yield of L-PAC by reaction C, especially under anaerobic conditions. Thus Bar's disclosure shows that the use of ACD reduces the yield of L-PAC. Surprisingly, and in sharp contrast to Bar's findings, we have found that the inclusion of beta-cyclodextrin (hereinafter often referred to as BCD) in the fermentation medium greatly increases the yield of L-PAC. Also surprising is our discovery that substantial improvements in L-PAC yield are obtained using quantities of BCD far less than the molar amount of benzaldehyde substrate added to the fermentation medium. It appears that conducting the bioconversion of benzaldehyde in the presence of BCD may serve to protect the yeast cells from toxic effects of the substrate and perhaps also of the product. Whatever its mode of action, it is clear that the BCD has an additional favorable effect of increasing bioconversion selectivity to the product L-PAC, in contrast to the findings of Bar who observed that ACD increased the selectivity to benzyl alcohol.
Thus the present invention provides a bioprocess that has greatly improved economics over the prior art in that it facilitates higher yields of the valuable product L-PAC from a substrate such as benzaldehyde that is expensive as well as toxic to the biological transforming system.